Department of Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, CT 06510; Department of Neuroscience, Yale University School of Medicine, New Haven, CT 06510; Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT 06510.

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Department of Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, CT 06510.

Department of Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, CT 06510; Department of Neuroscience, Yale University School of Medicine, New Haven, CT 06510; Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT 06510; elena.gracheva@yale.edu slav.bagriantsev@yale.edu.

Abstract

The ability to sense heat is crucial for survival. Increased heat tolerance may prove beneficial by conferring the ability to inhabit otherwise prohibitive ecological niches. This phenomenon is widespread and is found in both large and small animals. For example, ground squirrels and camels can tolerate temperatures more than 40 °C better than many other mammalian species, yet a molecular mechanism subserving this ability is unclear. Transient receptor potential vanilloid 1 (TRPV1) is a polymodal ion channel involved in the detection of noxious thermal and chemical stimuli by primary afferents of the somatosensory system. Here, we show that thirteen-lined ground squirrels (Ictidomys tridecemlineatus) and Bactrian camels (Camelus ferus) express TRPV1 orthologs with dramatically reduced temperature sensitivity. The loss of sensitivity is restricted to temperature and does not affect capsaicin or acid responses, thereby maintaining a role for TRPV1 as a detector of noxious chemical cues. We show that heat sensitivity can be reengineered in both TRPV1 orthologs by a single amino acid substitution in the N-terminal ankyrin-repeat domain. Conversely, reciprocal mutations suppress heat sensitivity of rat TRPV1, supporting functional conservation of the residues. Our studies suggest that squirrels and camels co-opt a common molecular strategy to adapt to hot environments by suppressing the efficiency of TRPV1-mediated heat detection at the level of somatosensory neurons. Such adaptation is possible because of the remarkable functional flexibility of the TRPV1 molecule, which can undergo profound tuning at the minimal cost of a single amino acid change.

Temperature sensitivity of dissociated neurons from dorsal root ganglia. Shown are baseline-normalized changes in intracellular Ca2+ recorded from the neurons in the zoomed insets of by ratiometric calcium imaging at different temperatures. Neurons were stimulated by heat ramp as shown on Top, followed by application of 1 μM capsaicin and 135 mM KCl solution as indicated by horizontal bars.

Alignment of amino acid sequences of rTRPV1 and sqTRPV1. Amino acid alignment of rTRPV1 (NP_114188), sqTRPV1 (KU877439), and caTRPV1 (XP_006172598) orthologs. SqTRPV1 and caTRPV1 are 89% identical to each other, and 90% and 85% identical to rTRPV1, respectively. The locations of ankyrin repeats (red boxes) and transmembrane domains (black bars) are depicted, along with the two amino acids identified in this study as being involved in temperature sensitivity (yellow boxes).

Voltage-clamp recordings of temperature and chemical responses of TRPV1 orthologs. Exemplar traces showing temperature- and capsaicin-evoked activity from control (uninjected) or TRPV1-expressing Xenopus oocytes measured at +80 mV and −80 mV by two-electrode voltage clamp. Oocytes were stimulated by a heat ramp followed by 5 μM capsaicin as indicated in Top.

Chemical and use-dependent modulation of TRPV1 thermosenstivity. (A) Proton concentration–response curves for TRPV1 orthologs in oocytes. Data shown as mean ± SEM (n ≥ 10) and fitted to a modified Hill equation. (B) Effect of pH on temperature activation as shown by heat responses of rTRPV1 and sqTRPV1 at pH 7.4 and 6.3. Recordings were normalized to maximum responses of each oocyte to 5 μM capsaicin (n ≥ 6). (C and D) Voltage-clamp recordings from oocytes in response to consecutive heat ramps. The second heat ramp was recorded in either standard physiological Ringer’s solution (C) or with the addition of 100 nM capsaicin (D). (E) Quantification of maximum current at +80mV and −80mV evoked by the second heat application (with or without 100 nM capsaicin) relative to the maximal response in the first heat ramp. Data shown as mean ± SEM from 7 to 18 oocytes. ***P < 0.001, ****P < 0.0001, unpaired t test.

Structure of the ankyrin repeat domain of TRPV1. The crystal structure of the ankyrin repeat domain of rat TRPV1 (PDB ID code: 2PNN) is shown along with the locations of the specific amino acids relevant to this study.